The long-term goal of the proposed research is to elucidate the molecular mechanisms by which FOXP transcription factors regulate specific gene expression in physiologically important processes. Mutations in FOXP proteins have been linked to human autoimmune disease (FOXP3) and speech disorder (FOXP2). Other members of the FOXP family have been implicated in tumor suppression (FOXP1) and lung development (FOXP4). Despite their significant physiological functions, the molecular mechanisms of this family of transcription factors are not well understood. FOXP contains a divergent forkhead domain and a number of distinct functional motifs, including a zinc finger/leucine zipper motif preceding the forkhead domain. There is also evidence that FOXP3 and NFAT1 physically interact and bind composite DNA sites cooperatively. Thus, either through intrinsic DNA binding or interaction with distinct partners, FOXP proteins possess unique DNA recognition mechanisms as compared classical FOX proteins, which in turn determine the transcriptional specificity of the FOXP family of transcription factors. The research proposed here will characterize the structure and function of FOXP proteins and their complexes with DNA and NFAT by X-ray crystallography and biochemical methods. Aim 1 will focus on the forkhead domains of FOXP proteins to characterize their detailed DNA recognition mechanisms. Aim 2 will analyze the structure of the zinc finger/leucine zipper motif and larger fragments or full-length FOXP proteins to see how other regions of FOXP modulate the function of this family of proteins. Aim 3 will study the structural basis of NFAT/FOXP interaction and its potential function in T cell gene expression. The detailed biochemical and structural studies proposed in this application will provide a molecular basis for analyzing the mechanisms of disease- associated mutations in FOXP proteins and further investigation of their normal physiological roles in diverse cellular processes. Health Relevance: Because mutations in FOXP proteins are linked to autoimmune disease and speech disorder, uncovering their unique DNA recognition mechanisms will help identify potential target genes of these transcription factors, which in turn can offer crucial insights into the mechanisms of human language development and immunological tolerance. These insights may lead to new approaches to diagnosing and treating human diseases incurred by malfunction of FOXP proteins.